1. Field of the Invention
This invention is directed to a system, test method and test strip for electrochemical quantitative analysis of one or more analytes in a test sample. More specifically, this invention is directed to an electrochemical quantitative analysis system for determination of an analyte (e.g. proteins, hormones or enzymes, small molecules, polysaccharides, antibodies, nucleic acids, drugs, toxins, viruses or virus particles, portions of a cell wall and other compounds which have specific or characteristic markers that permit their identification) within a solid phase (e.g. test strip) by measurement of electrochemical changes of label(s) associated with a labeled substance that can be correlated with the concentration of the analyte of interest. The test strip used in this analytical system includes a solid phase affinity chromatographic medium, having an electrochemical detection system associated therewith, within the fluid pathway through said medium. The inventions hereinafter set forth in detail are applicable to quantitative analysis of virtually any analyte, whose concentration can be correlated with an electroactive species of an electroactive substance that is capable of manifesting a change in electrical response within the electrochemical cell-like environment of the test strip of this invention.
2. Description of Prior Art
A. Immunodiagnostics
Immunochemical analysis for an analyte within a solid phase (test strip) generally involves an assay in which an analyte, in a liquid sample, and a labeled substance, interact with each other, or another reaction constituent. Within the context of the prior art, it is understood that a xe2x80x9clabeled substancexe2x80x9d can, thus, include (a) an analyte mimic that is labeled with an indicator, (b) a labeled ligand that is specific for interaction with the analyte of interest, or (c) a complex that is formed by interaction of (a) or (b) and yet another reaction constituent. The liquid containing the labeled substance and the analyte is then transferred to a porous film or membrane, where it is drawn by diffusion or capillary action along a fluid pathway of the membrane, and encounters one or more companion test kit reagents (at least one of which is immobilized within a definitive area thereof). This labeled substance and/or the analyte is thereafter captured by an immobilized companion test kit reagent at a test site to form a complex. If the complex is present in sufficient concentration, it produces a discernible change at such site. Where the indicator is a pigment, such as a metal (e.g. colloidal gold), or alternatively, a colored latex particle, the discernible change is generally visible to the naked eye.
The following patents are regarded as representative of this type of immunochromatographic assay. These patents are arranged in chronological order and no significance is to be attached to their order of discussion as to the patentability of the invention described and claimed herein.
Campbell et al, U.S. Pat. No. 4,703,017 (assigned to Becton Dickinson) describes a test method for detection of an analyte with a xe2x80x9cdirectxe2x80x9d or xe2x80x9cparticulatexe2x80x9d indicator, specifically an indicator that can be visually detected with the naked eye, thus, making such test method acceptable for home diagnostic use; or, alternatively, in a clinical setting devoid of sophisticated analytical equipment. The direct indicator of choice is a liposome containing a pigment or equivalent colorant. The claimed test method is described as suitable for urine analysis of analytes (e.g. pregnancy test); and, thus, capable of detection of an elevated level of analyte. Accordingly, such assay is regarded as a semi-quantitative determination of the analyte of interest. Because the test involves a single step (e.g. application of the sample to the test strip), the test has become characterized in the art as a xe2x80x9cRapidxe2x80x9d test.
Rosenstein, U.S. Pat. No. 5,591,645 (assigned to Becton Dickinson) describes an immunochromatographic test method based upon the principles described in commonly assigned U.S. Pat. No. 4,703,017 (to Campbell et al). The Rosenstein test device incorporates all of the test kit reagent in the xe2x80x9csame planexe2x80x9d of the test strip and contemplates applying the sample directly to the test strip to reconstitute the reagents contained therein and, thereby effect the desired analysis. The direct indicator of choice can be selected from a number of equivalent colorants, however, colloidal gold adsorbed to a binding protein (e.g. antibody or antigen) is generally preferred. The claimed test method is described as suitable for urine analysis of analytes (e.g. pregnancy test); and, thus, capable of detection of an elevated level of analyte or a semi-quantitative determination of the analyte of interest. Because the test also involves a single step (e.g. application of the sample to the test strip), the test has become characterized in the art as a xe2x80x9cRapidxe2x80x9d test.
Swanson et al, U.S. Pat. No. 5,073,484 (assigned to Abbott laboratories) describes an immunochromatographic test method utilizing multiple test zones, with each zone arranged along a common linear fluid pathway. As the test sample and test kit reagents migrate along this fluid pathway, the analyte of interest (generally associated with a direct indicator) becomes bound to an immobilized companion reagent in each of the test zones. Each of the test zones is precalibrated in reference to a standard, and thus the appearance of color in specific test zone correlated with the concentration level of analyte in the sample. Accordingly, the concentration of the analyte in the sample can be estimated from the level of color development along the fluid pathway of the test device. Because the test also involves a single step (e.g. application of the sample to the test strip), the test has become characterized in the art as a xe2x80x9cRapidxe2x80x9d test.
May et al, U.S. Pat. No. 5,602,040 (assigned to Unilever Patent Holdings) describes an immunochromatographic test method analogous to the test methods described in the Campbell et al and Rosenstein patents discussed above. One of principles differences identified and claimed by May et al, is the lyophilization of a sugar, along with the direct indicator, in the sample receiving site. The sugar addition is reportedly required to effect reconstitution of the lyophilized indicator and thus permit its interaction with the analyte in the sample.
With the limited exception of the adaptation of the foregoing Rapid Tests to detection of an elevated level of analyte, these Rapid Tests are generally unsuitable for precise quantitative monitoring an analyte level, or for the quantitative detection and differentiation of more than one analyte within a single test environment.
B. Instrumental Analysis
Analysis of fluid samples has also traditionally involved the use of instrumentation and the measurement of changes in electrical potential and/or current at given site/electrode. Typically, the electrical measurement is referenced to a second electrode, and a number of measurements made over a given interval and/or defined range of conditions.
The following patents are regarded as representative of this type of electrochemical analysis. These patents are arranged in chronological order and no significance is to attach to the order of discussion, or to their relative significance to the patentability of the invention described and claimed herein.
Wang U.S. Pat. No. 5,292,423 (assigned to New Mexico State University Technology Transfer Corp.) describes a method and apparatus for trace metal analysis of fluid samples (e.g. drinking water, blood, urine etc.) by means of initial adsorption of the trace metal from a fluid sample onto a screen printed carbon (working) electrode that had been pre-coated with mercury. The Wang test platform also employs at least one additional (reference) electrode. The electrode having the trace metal was, thereafter, subjected to analysis by either Anodic Stripping Voltammetry (ASV) or Potentiometric Stripping Analysis (PSA). The reported advantages of the Wang method and apparatus include the ability to adapt his invention to on-site field testing of sample fluids for suspected pollutants with a disposable testing device.
Brooks U.S. Pat. No. 5,753,517 (assigned to the University of British Columbia) describes a quantitative immunochromatographic assay utilizing an apparatus for detection of a particulate latex indicator in accordance with the procedures associated with so called RAMP(trademark) technology. According to Brooks et al., the RAMP(trademark) technology utilizes latex particles in a manner similar to enzymes in ELISA assays. In the RAMP(trademark) technology based analysis, a test strip, having two distinct fluorescent labeled indicators, is placed in a cartridge, and a fluid sample applied to the test strip. The analyte of interest interacts with one of the indicators forming a fluorescent complex which is captured in the test zone of the test strip, and while the second indicator remains in tact within the test strip, (so as to provide an internal standard). The RAMP(trademark) technology is, thus, capable of differentiation of the fluorescent complex from the internal standard, and the amount of analyte in the sample determined thereby.
Brooks et al. alternative system for performance of his analysis, contemplate the use of optical detection methods (light scattering) and measurement of changes in electrical conductivity or resistively. In one of the suggested alternatives, Brooks et al. contemplates (U.S. Pat. No. 5,753,517xe2x80x94col. 7, lines 7-19, inclusive) the quantitative measurement of the analyte of interest by the electrochemical detection of a released electroactive agents, such as bismuth, gallium or tellurium ions, from a complex associated with the analyte of interest. According to Brooks et al one of these alternatives involves the use of a chelating agent-protein conjugate as an indicator for the analyte of interest, and detection thereof contemplates the addition of an acidic solution for release the metal label as ions for later quantitation by anodic stripping voltammetry (as described by Hayes et al, Anal. Chem. 66:1860-1865 (1994).
The following published technical literature is regarded as representative of this type of electrochemical analysis. These papers are arranged in chronological order and no significance is to attach to their order of discussion, or to their relative significance to the patentability of the invention described and claimed herein.
Electrochemical techniques have provided challenges associated with affinity-chromatographic reactions, because of their stable and sensitive signal, lower level of detection limit, simple operation and cost-effectiveness. Pioneering studies by Henieman et al.(Hayes, F. H. et al., Anal. Chem., 66, 1860-1865 (1994)) illustrated the use of metal ion labels for heterogeneous immunoassays with Anodic Stripping Voltammetric (ASV) detection. Such immunoassays involved covalently linking chelating agent to a protein to serve as a chelon for the metal label. Following competitive equilibrium between the labeled and unlabeled protein for the antibody (immobilized on the surface of a polyester tube), the metal label was released and transferred to an electrochemical cell for an ASV detection with a hanging mercury drop electrode (HMDE) and a deaerated solution. Similarly, Wang et al. (Wang J., Anal. Chem., 70, 1682-1685 (1998)) employed an antibody-coated, screen-printed sensor, performed the entire assay protocol directly on the surface of the disposable strip, and employed the highly sensitive potentiometric stripping mode for detecting the released metal ion label in microliter solutions. As desired for decentralized sensing applications, such an on-chip protocol offers several advantages compared to conventional ASV-based immunoassays, including simplified operation (e.g., the elimination of the separation and reagent volumes, elimination of toxic mercury drops, and a more sensitive stripping detection mode). The Wang et al. system requires relatively long incubation periods for pre-conditioning the sensor (prior to use) and for interaction with the sample; and, multiple washing steps, both of which make this method impractical and cumbersome.
It is the object of this invention to remedy the above and related deficiencies in the prior art.
More specifically, it is the principle object of this invention to adapt rapid affinity chromatographic electrochemical analysis to quantitative determination of analytes of interest.
It is another object of this invention to provide a system for electrochemical quantitative analysis of an analyte(s) within a solid phase test format that is comparable in ease of use to rapid immuchromatographic assays.
It is yet another object of this invention to provide a system for electrochemical quantitative analysis of an analyte(s) within a solid phase test format involving a simplified means for detection of labels by potentiostatic or potentiometric measurement of changes within the solid phase that are attributable to the concentration(s) of label(s) within the test site.
It is still yet another object of this invention to provide a system for concurrent electrochemical quantitative analysis of a common test sample for multiple analytes within a solid phase test format involving a simplified means for detection of label(s) by stripping voltammetry
The above and related objects are achieved with the electrochemical quantitative analysis system, method and test strip of this invention for determination of the concentration of an analyte within a solid phase test environment. Initially, a test sample solution and a labeled substance are initially contacted, under assay conditions, within a solid phase test environment, and caused to migrate along a fluid pathway therein. Irrespective of the assay format (competitive assay, sandwich assay, etc.), the labeled substance is concentrated (bound) within a delimited area of the solid phase. Within the context of the system and analytical method of this invention, a xe2x80x9clabeled substancexe2x80x9d can include any suitable electroactive material that can be isolated within a delimited area of the solid phase, under assay conditions, or which can release an electroactive component within such delimited area, (also collectively hereinafter referred to as the xe2x80x9celectroactive speciesxe2x80x9d); and, such electroactive species thereafter, undergo an electrochemical transition (e.g. redox) incidental to electrochemical quantitative analysis. The observed measurement (transition) of the electroactive species can be directly or inversely related, (e.g. by comparison to a standard curve), to the concentration of the analyte of interest in the test sample.
The two principal types of electroanalytical measurements for determination of the concentration of the analyte of interest are potentiometric and potentiostatic. In each of these analytical protocols, two electrodes are required/involved, and a contacting sample (electrolyte) solution, which together constitute an electrochemical cell-like environment within the test strip. The electrode surface is, thus, the junction between the ionic conductor (e.g. electrolytes from the aqueous fluid sample, test kit reagents, etc.) and an electronic conductor. Within this electrochemical cell-like environment of the test strip, the electrode that responds to the analyte of interest (or an electroactive substance that is indicative of the analyte of interest), is termed the xe2x80x9cindicatorxe2x80x9d or xe2x80x9cworkingxe2x80x9d electrode, whereas the electrode that is maintained at a constant potential is termed the xe2x80x9creferencexe2x80x9d electrode (its response being independent of the sample solution).
In one of the embodiments of this invention, the immobilized labeled substance is contacted with a release reagent to initially cause the release/displacement of the label from the immobilized labeled substance within this delimited area. In the case of a metal label, for example, the released/displaced metal can be further reacted with mercuric ions in the release reagent solution, to form an amalgam on the surface of the working electrode within the delimited area of the solid phase. Thereafter, the delimited area of the affinity chromatographic test strip is subjected to potentiostatic measurement, by anodic stripping voltammetry of the label from the amalgam on the working electrode, which causes the label, to undergo yet a second electrochemical transition, (conversion of the label from the reduced form to the oxidized state). This second electrochemical transition of the label (from the reduced to the oxidized state) has a characteristic fingerprint that can be monitored and which, when compared to a standard curve, can correlate directly with the concentration of the analyte in the sample.
The stripped label from the amalgam and, thus, the analyte of interest, can be quantified by measurement of changes within the delimited area of the test strip by potentiostatic electrochemical techniques. The specifics of this electrochemical quantitative analysis, involve the application of an electrical potential to the delimited area, over a defined range of potentials, and the monitoring the rate of electron transfer (current) at each potential. The variation of electrical potential is analogous to the taking of a series of optical measurement of a colored indicator at varying the wavelengths. The electrical potential applied to the electrode drives (forces) the targeted electroactive species to gain or lose electrons (reduction or oxidation, respectively) at a given rate that is indicative of the concentration of such electroactive chemical species. Accordingly, the resultant current not only reflects the rate at which the electrons move across the electron/solution interface, but also (when compared to a standard), the concentration of the analyte in the test sample. These potentiostatic techniques can, thus, measure any chemical species that is electroactive, (e.g. that can be made to reduce or oxidize within the environment of an electrochemical cell)
One of the preferred designs of the test strip useful in the electrochemical quantitative analysis system of this invention has multiple components and multiple functional areas to (a) control the volume and rate of absorption of sample by the test strip; (b) accommodate the controlled interaction of the sample and a labeled substance with an immobilized binding substance within a delimited area of the test strip; (c) effect the separation of the labeled substance from the endogenous components of the sample; (d), the concentration of the labeled substance within a delimited area of the test strip for electrochemical processing; and, (e) the quantitative determination of the analyte of interest by electrochemical means.
In another of the preferred embodiments of the foregoing test strip design, the sample and the labeled substance can be combined within a bibulous pad (e.g., fiberglass) that is maintained in fluid communication with a solid phase affinity chromatographic test medium. Upon transfer of the sample thereto, the labeled substance is reconstituted, and the resultant labeled substance and/or a complex thereof which is formed with the analyte of interest, drawn into such test medium. As this fluid and its constituents, (e.g. sample, test kit reagent and reaction products thereof), diffuse into and within the affinity chromatographic test medium, the labeled substance becomes bound to a delimited area (also hereinafter xe2x80x9ctest sitexe2x80x9d) along this fluid pathway. Typically, this delimited area can be defined by means of an immobilized binding substance that is specific for interaction with the analyte, an analyte mimic or a complex of the analyte and/or a labeled ligand (e.g. by binding to an epitope on the analyte). As the analyte, analyte mimic or a complex of the analyte and/or a labeled ligand becomes increasingly concentrated at the test site, it can be measured and the amount thereof correlated with the analyte in the test sample.
The advantages of such controlled potential (potentiostatic) techniques include high sensitivity, selectively toward an electroactive species, a wide linear range, portable and low cost instrumentation and speciation capability.
In order to accommodate multiple functional components within the integral device of the preferred test strip design of this invention, all of the components are preferably arranged/mounted on a common support or backing layer (typically an inert plastic, e.g. Mylar). The substrate of this test device can be pre-printed with a conductive material to afford electrical contact (direct or inductive coupling) between the affinity chromatographic test medium of the test strip and an electrochemical analyzer that is configured to measure subtle electrical changes within the delimited areas in the test strip. As more fully set forth in the description of the Figures, which follows, this support layer is typically imprinted at two or more locations with a conductive material or metal salt, to form what are referred to hereinafter as xe2x80x9celectrodesxe2x80x9d. These electrodes are spatially arranged along the support layer to coincide with delimited areas of the solid phase medium. The electrode coincident with the delimited area of the test site is referred to as the xe2x80x9cworking electrodexe2x80x9d. Depending upon the electrochemical method of analysis, the test strip generally requires at least one additional (e.g. xe2x80x9creferencexe2x80x9d electrode) to form the plates of an electrochemical cell.
After having concentrated the labeled substance within the delimited area of the test site, it can be measured by a number of electrochemical techniques. As noted above, it may be desirable, preliminary to performing such measurement, to first isolate the label from the complex by xe2x80x9cpre-concentrationxe2x80x9d thereof on the working electrode. This xe2x80x9cpre-concentrationxe2x80x9d process can involve the release/displacement of the label from the complex and the capture of the label in a reduced form (e.g. amalgam) on the working electrode where it is more electrochemically available or active. Upon completion of this pre-concentration process, the resultant amalgam can be subjected to potentiostatic electrochemical quantitative analysis by anodic stripping voltammetry. In the context of this preferred analytical system (e.g. potentiostatic electrochemical quantitative analysis by anodic stripping voltammetry), the label is reoxidized, under electrochemical quantitative analytical conditions, and this electrochemical transition of the label (from the reduced to the oxidized state) is monitored. The current signal that is generated during this transition (peak and area) is dependent upon a number of system variables, the characteristics of the metal label and the electrode geometry. In each instance, however, these variables can be optimized by empirical adjustment; and, the analysis conditions tailored to correspond to standard curves which are used to correlate electrical signal response to analyte concentration. More specifically, FIG. 3 is a graphical depiction of current signal response of a bismuth/mercury amalgam on a carbon electrode to anodic stripping analysis over a range of concentrations (1 ng/ml to 50 ng/ml of pre-concentrated bismuth metal label). Similarly, FIG. 4 illustrates the variation in signal (current) intensity, as a function of the duration of xe2x80x9cpre-concentrationxe2x80x9d interval, (prior to electrochemical analysis). In the graphical depiction of this variable shown in FIG. 4, the longer the pre-concentration interval (which varied from 1 to 15 minutes) the greater the current signal intensity. Similar correlation/optimization in analytical process conditions is made for each of the labels that are selected and standard curves for each created, as appropriate, to correlate with the dynamic range of conditions likely to be encountered for a given analyte and over a range of concentration.